Sandra Alves -INSArepositorio.insa.pt/bitstream/10400.18/2856/1/Jornadas...Project FCT:...

LysosomalLysosomal Storage Diseases: Storage Diseases: pathophysiologypathophysiology and innovative therapeutic approachesand innovative therapeutic approaches

Jornadas CECA 2014

Inborn Errors of Metabolism in animals / natural models subunit

LysosomalLysosomal Storage Diseases: Storage Diseases: pathophysiologypathophysiology and innovative therapeutic approachesand innovative therapeutic approaches

G>A

UID - Departamento de Genética Humana

Sandra Alves - INSA

Sandra Alves, Olga Amaral, Francisca Coutinho, Liliana Matos, Ana Joana Duarte, Diogo Ribeiro.

Group members

- Maria João Prata - IPATIMUP.

- Belén Pérez. Centro de Biología Molecular Severo Ochoa Nicolas Cabrera, Universidad Autónoma de Madrid.

- Daniel Grinberg. Centro de investigação Biomédica em Rede de Doenças Raras (CIBERER). Barcelona.

- Thomas Braulke, Department of Biochemistry, University Medical Center Hamburg-Eppendorf.

- Alexey Pshezhetsky - CHU Sainte-Justine, Montreal, Canada

Colaborations:

Lysosome – central role in cell metabolism

The biochemical and cellular basis of lysosomal storage disorders

Disease Defective protein Main storage materials

Clinical Symptoms

Diagnosis

Lysosomal Disorders and Common Therapeutic Approaches

MolecularMolecular andand cellularcellular characterizationcharacterization ofof lysosomallysosomal diseasesdiseases:: diversity,diversity, frequencyfrequency andand causalitycausality

•“Prognosis and prevention in a few inherited diseases existing in the Portuguese population”

PIC/IC/82822/2007.

• “Molecular analysis of the mucolipidosis II and III in Portugal: characterization of the mutational

spectrum and relationship with clinical phenotypes”. PIC/IC/83252/2007

•“The sorting and trafficking of lysosomal proteins through M6P independent pathways: molecular,

biochemical and functional studies”. PTDC/SAL-GMG/102889/2008

DevelopmentDevelopment ofof alternativealternative therapeutictherapeutic approachesapproaches

• “Splicing therapeutics for patients affected by a lysossomal storage disorders”. (FCT

Research Lines/Projects

• “Splicing therapeutics for patients affected by a lysossomal storage disorders”. (FCT

SFRH/BD/64592/2009). ”

• Development of a U1 snRNA-adapted gene therapeutic strategy to correct 5’ splicing defects in

lysosomal storage disorders”. SPDM/Genzyme Grant.

• Less is more - Substrate Reduction Therapy for Mucopolysaccharidoses through RNAi. Fundação

Millennium bcp grant.

• Cost action BM1207 – Networking towards application of antisense mediated exon skipping (2013-

2017).

OtherOther projectsprojects

• Use of next generation sequencing, for the study of patients presenting symptoms frequently

associated with LSDs, but without a specific diagnosis.

• Development of iPSCs as disease specific cellular models for LSDs

Molecular and cellular characterization of lysosomal diseases: diversity, frequency and causality

Mucopolysaccharidoses type II

16 16 unrelated Portuguese patients;

1515 different mutations;

9 of the 15 affected the usual splicing pattern splicing pattern at

the locus.

Functional analysis of IDS splicing defects

IDS gene presents alternative splicing: 11 different transcripts

Complex underlying regulatory mechanism that probably requires the interaction of multiple

splicing factors, and make this gene prone to the occurrence of mutations that disrupt splicing

c.257C>T and c.241C>T (exon 3, IDS gene)

splicing factors, and make this gene prone to the occurrence of mutations that disrupt splicing

Through overexpression or depletion assays, we experimentally demonstrated that SC35

and hnRNP E1 proteins are involved respectively, in the use and repression of the

constitutive 3’ splice-site of exon 3.

Matos L et al. (2014) under preparation


Mucopolysaccharidoses type IIIA e IIIB

1111 Portuguese patients;

55 novel mutations.

Frequent mutation: R234C (also in Spanish patients)

Haplotypic analysis: sharing of the an ancestral

haplotype by Portuguese and Spanish patients - a haplotype by Portuguese and Spanish patients - a

common origin of the mutation in Iberia (recent).

Mucopolysaccharidoses type IIIC

33 patients;

22 novel mutations.

Sialidosis Galactosialidosis and Glangliosidosis type 1

2121 Portuguese patients studied:

33 affected with sialidosissialidosis (NEU1) ,

44 with GalactosialidosisGalactosialidosis (PPGB) and

1414 with GlangliosidosisGlangliosidosis type 1 type 1 (GLB1)

Several novel missense mutations were reported


Several novel missense mutations were reported

Mucolipidosis type II and III

. A set of 2323 unrelated ML II and III cases from several origins was screened:

1818 different mutations → 1616 in the GNPTG gene and 22 in the GNPTG gene.

Of those, 13 were novel: 11 in the GNPTAB gene and 2 in the GNPTG gene.

• Identification of the first largelarge deletiondeletion in the GNPTAB gene


Origin and spread of a common deletion c.3503_3504delTC

44 44 patients and 16 16 carriers from different geographic

regions were analyzed for

33 intragenic polymorphisms and

22 microsatellite markers flanking the GNPTAB

gene.

A common haplotype was identified in all chromososmes

bearing the deletion: common origin common origin (aprox. 2000 years ago)

Its geographical distribution also suggested it to have

arisen in the periperi--Mediterranean regionMediterranean region.

Nonsense/ FrameshiftNonsense/ Frameshift MisseMissensense

MissenseMissense and frameshiftframeshift mutations that

are associated with a severe clinical

phenotype cause retention of the

encoded protein in the endoplasmic

reticulum in its precursor form


reticulum in its precursor form

120 individuals with clinical suspicion of LSD but

without definitive biochemical and/or molecular

diagnosis,

nono novelnovel mutationsmutations were detected either on the

SCARB2 or on the SORT1 genes.

SCARB2

SORT1

LSD patients without specific diagnosis

Molecular screening of genes involved in mannose

6-phosphate independent trafficking

Molecular and biochemical characterization of LSDs patients and pathophysiological mechanisms

SCARB2 mutations as phenotypic modifiers in Gaucher disease

To evaluate the role of SCARB2 mutations on the Gaucher

Disease phenotype, the whole cohort of Portuguese GD

patients (which totalizes 91 individuals) was screened and 1

novel mutation in the SCARB2 gene identified.

T398M

SCARB2

Coutinho MF (2014) under preparation

Unverricht- Lundborg

Unverricht- Lundborg disease (ULD ou EPM1, MIM 254800), is a myoclonic epilepsy,

caused by mutations in the CSTB gene which lead to impaired function of cystatin B and

failure in the inhibition of lysosomal proteases.

•Identification of a rare molecular mechanism causal of Unverricht Lundborg disease in

a unique Portuguese patient.


Project FCT: PIC/IC/82822/2007 (2010-2013), IR: Olga Amaral

Unverricht-Lundborg disease is caused by mutations in the cystatin B gene

compromising its protective anti-protease function. The normal protein has

several cellular localizations, it is found in the nucleus, cytosol and lysosome.

A B C

CT P

Cystatin B (A-12)

~ 12 KDa

Fig 1: Western analysis of

Molecular and cellular characterization of normal and mutant cystatin B


• What is the pathogenic mechanism involved in this progressive epilepsy?

• What is the extent of lysosomal involvement?

• In patient fibroblasts the protein quantity is clearly reduced (Fig.1 and Fig.2)

although its location seems to be preserved. Could there be differences in other

cell types?

CSTB: CSTB:

A) control; B) patient; C) CSTB/LAMP:patient

Fig 1: Western analysis of

skin fibroblasts of control

and patient

Fig 2: IF study in skin fibroblasts with specific antibodies


Mucopolysaccharidosis type I (MPS I) results from the defective

activity of the lysosomal α-L-iduronidase enzyme (IDUA; EC 3.2.1.76).

MPS I is one of the more frequent MPSs. In the Portuguese

population it is often associated with mutation W402X (over 60% in

unrelated patients).

Caracterization of the IDUA gene mutation W402X (Diogo Ribeiro, MSc)

•Mutation W402X results


63% 60%45%

37%

11%4%

79%

37%

0%10%20%30%40%50%60%70%80%90%

100%

Fre

qu

ên

cia

alé

lica

d a

mu

taçã

o W

40

2X

em

do

en

tesM

PS

I

•Mutation W402X results

in NMD, seems amenable

to correction by nonsense

suppression.

•Presence of different

haplotypes in Portugal

Project FCT:

PIC/IC/82822/2007 (2010-

2013), IR: Olga Amaral


- Development of low cost mutation detection techniques (Diogo Ribeiro et al. 2011; 2013)

- Identification of rare variants which influence prognosis (Ana Duarte et al. 2013; Amaral et al. 2013; Rodrigues et al, 2004)

- Clarifying molecular basis of Lysosomal Diseases (Amaral, et al. 1997, 1999 & 2000; Ribeiro, et al. 2001; Pinto et al. 2010)

- Prevalence of various disease causing mutations in

different populations (Amaral, et al. 1994; Marcao, et al. 1999; Pinto et al. 2004; Lugowska et al. 2005; Cherif et al. 2009 & 2013)


Use of next-generation-sequencing (NGS) as an approach for the

diagnosis of LSDs

Future

plans

To develop a next generation sequencing (NGS) based workflow for the

identification of exon-variations in a group of genes involved in lysosomal

function.

Aim


Uncharacterized patients presenting symptoms associated with LSDs

The use of next generation sequencing in this group of patients with suspected LSD

but no definitive diagnosis would be the ideal approach to search for the genetic

basis of disease in these individuals

In the scope of Comissão Coordenadora do Tratamento das Doenças

Lisossomais de Sobrecarga (CCTDLS) that operates under INSA

coordination - Despacho n.º 2545/2013

Target

U1 snRNA-mediated Gene Therapeutic Approach in ULD patient-derived fibroblasts.

Control (2.5 µg) Patient (2.5 µg)

MU

T

WT

TC

MU

T

WT

TC

Unverricht- Lundborg disease - c.66G>A (CSTB gene)

Splicing Therapeutics - antisense oligonucleotide and U1snRNA mediated

therapeutic strategies

Development of innovative therapies for LSDs

U1-CSTB-wt

U1-CSTB-tc

U1 WT

WT splice donor siteCAGgugggtgg…

GUCcauucaua

Exon 15’

3’

U1-CSTB-mut

5’…CAAgugggtgg…

3’…GUTcauucaua…

3’…GUCcacccacc…

3’…GUTcacccacc…

Mutant splice donor site

M NC U1

WT

500 bp

100 bp

NT U1

-CS

TB

-MU

T

U1

-CS

TB

-WT

U1

-CS

TB

-TC

NT U1

WT

U1

-CS

TB

-MU

T

U1

-CS

TB

-WT

U1

-CS

TB

-TC

1 2

21 354 bp

Intron 1

Matos et al. under preparation

No correction

5’ AGCCGGCTGCTCACCTGCGCCATCGCCG 3’

Exon 1 Intron 1 Exon 2354 bp


Antisense Oligonucleotide Therapeutic Approach in ULD patient-derived fibroblasts.

Unverricht- Lundborg disease - c.66G>A (CSTB gene)

Splicing Therapeutics - antisense oligonucleotide and U1snRNA mediated


M NC HC 5

ULD patient (LNA nM)

10025 500

500 bp

100 bp

1 2

21 354 bp

Intron 1

Anti - Cystatin B

ULD Patient

(LNA nM)

Control

(LNA nM)

1000

Anti - α-Tubulin

11 kD

50 kD

0

Correct splicing

354 bp

Successful correction of the aberrant transcript


Splicing Therapeutics - Antisense oligonucleotide and U1snRNA mediated



Antisense Oligonucleotides treatment of

patient’s fibroblasts

M B CT DT 30 5050 100

AMO (µM) LNA (nM)

500 pb

Mucopolysaccharidosis type II c.1122C>T (IDS gene)

No correction

Development of antisense therapeutic approaches remains

a challenge in genes under fine regulation mechanisms as

is the case of IDS gene.


Mucopolysaccharidosis type IIIC c.234+1G>A (HGSNAT gene)

A partial recovery was achieved with a modifiedU1 snRNA that completely matches the splice

donor site.

Min

i W

T e

x2

U1

WT

U1

su

p 1

U1

su

p 2

U1

su

p 3

U1

su

p 4

Mini WT ex2

U1 snRNA-mediated Gene Therapeutic Approach in MPS IIIC patient-derived fibroblasts.

Splicing Therapeutics - Antisense oligonucleotide and U1snRNA mediated



U1 wt

HGSNAT exon 2:

wt splice donor siteCACguauguau…

GUCcauucaua

Exon 2

Splice donor site

5’

3’

HGSNAT exon 2: mutated splice donor site

(c.234+1G>A)5’…CACauauguau…

3’…GUC tauucaua…

3’…GUGtauucaua…

U1 sup 1 (+1T)

U1 sup 2 (-1G +1T)

3’…GUGcauacaua… U1 sup 3 (-1G +4A)

3’…GUGtauacaua… U1 sup 4 (-1G +1T +4A)

Exon 2V V

V V

C-M Min

i W

T e

x2

U1

WT

U1

su

p 1

U1

su

p 2

U1

su

p 3

U1

su

p 4

500 bp

200 bp

NT

V VInt 2

4ntExon 2V V

Min

i W

T e

x2

U1

WT

U1

su

p 1

U1

su

p 2

U1

su

p 3

U1

su

p 4

Mini mut ex2

C-M Min

i m

ut

ex2

500 bp

200 bp

NT

Matos et al. submitted

Mutations under study:

Mucopolysaccharidosis I: c.1650+5G>A (IDUA gene)

Mucolipidosis III: c.3335+6T>G (GNPTAB gene)


3 U1 variants were constructed for c.1650+5G>A

2 U1 mutant vectors were constructed c.3335+6T>G

transfected using Lipofectamine LTX

No correction

Splicing Therapeutics - Antisense oligonucleotide and U1snRNA mediated therapeutic

strategies

Novel approach: U1 lentivirus plasmid will be used to generate through mutagenesis

the same U1 mutant vectors that will be used in a model cell line with mutation-

disease minigenes as well as directly in patient’s fibroblasts through the transduction

lentiviral technique.

No correction

Substrate Reduction Therapy for Mucopolysaccharidoses through RNAi


RNAi-dependent strategy based

Selective downregulation of genes involved in the

biosynthesis of the glycosaminoglycans (GAGs)

that accumulate in patients suffering from that accumulate in patients suffering from

Mucopolysaccharidoses.

WorkPlan

• Design specific siRNAs targeting genes involved in the biosynthesis of

GAGs.

– Targets:

• CHSY1, CHPF e CHSY3;

• EXTL2 e EXTL3;

• XYLT1,XYLT2,GALTI, e GALTII


Substrate Reduction Therapy for Mucopolysaccharidoses through RNAi

• XYLT1,XYLT2,GALTI, e GALTII

• Evaluate the effect of each specific siRNA in the

mRNA expression levels and in its target protein

expression levels.

• Evaluate the effect of each specific siRNA and/or

combination of individual siRNAs on the

intralysosomal accumulation of GAGs in MPS

patients’ cell lines.

mRNA

GAGs

Future directions...

• Design a proper siRNA delivery strategy for therapeutic purposes


• In vivo evaluation in animal models.

These experiments will greatly benefit with

our integration in CECA.

Future directions: generation of iPSCells as disease specific cellular models for LSDs

Figures: Grskovic et al, 2011, Nat Rev Drug Discovery, 10:915-929

Generation of disease specific cell-targets

CECA might provide a favorable environment

for fostering part of this work

Future directions : iPSCs as disease specific cellular models for LSDs

Figure from: Parenti et al, 2013, International J Molecular Med, 31(1), 11-20

Date post:	08-Feb-2021
Category:	Documents
Upload:	others
View:	1 times
Download:	0 times

Sandra Alves -INSArepositorio.insa.pt/bitstream/10400.18/2856/1/Jornadas...Project FCT:...

Documents